HIV-1 Vpr plays a pivotal role in viral pathogenesis, as its functions are being linked to nuclear transport of visual pre-integration complex, viral replication and suppression of human immune function. However, little is known about the molecular mechanisms underlying these effects. In this proposal, we will focus on studying two related effects of Bpr on cell cycle G2/M control and proteolysis, which will help us to further understand the molecular basis of these viral effects on the host cellular functions. We have successfully accomplished three proposed Specific Aims. 1) to define the functional domains of Vpr responsible for nuclear localization, G2 arrest and cell killing, 2) to identify the cellular pathways affected by Vpr when it interrupts the cell cycle, and 3) to investigate the specific roles of PP2A in Vpr-induced G2 arrest. We showed that Vpr activities in fission yeast cells are very similar to those in mammalian cells. We also found that Vpr does not induce G2 arrest through the two classic DNA damage or replication checkpoints but instead through a PP2A-dependent novel regulatory pathway. In addition, we have identified a number of genes which when over- expressed suppress the G2 arrest and nuclear localization of Vpr, and these suppressions suggest a new role for Vpr in the regulation of proteolysis. For the proposed studies, we hypothesize that Vpr induces G2 arrest through a novel PP2A-dependent regulatory pathway(s), and Vpr affects proteolysis by interaction with the proteasome on the nuclear periphery. Three new specific aims are proposed to test these hypotheses. 1) To define and characterize the cellular components of the new PP2A- dependent regulatory pathway by which Vpr induces G2 arrest. 2) To investigate the potential interaction of Vpr with the proteasome at the nuclear periphery. 3) To evaluate the possible role of pr-proteasome interaction in the regulation of proteolysis including a relationship between Vpr-induced G2 arrest and proteasome activity. These proposed studies will be confirmed in human cells and provide important new insights into fundamental aspects of the Vpr's effects on these two basic cellular functions.